Glycosylation is the covalent linkage of an oligosaccharide chain to a protein resulting in a glycoprotein. In many glycoproteins, the oligosaccharide chain is attached to the amide nitrogen of an asparagine (Asn) residue and leads to N-glycosylation. Glycosylation represents the most widespread post-translational modification found in natural and biopharmaceutical proteins. It is estimated that more than half of the human proteins are glycosylated and their function frequently depends on particular glycoforms (glycans), which can affect their plasma half life, tissue targeting or even their biological activity. Similarly, more than one-third of approved biopharmaceuticals are glycoproteins and both their function and efficiency are affected by the presence and composition of their N-glycans. Leafy crops, such as the tobacco plant Nicotiana benthamiana, are an attractive system for the production of therapeutic proteins, as plants are generally considered to have several advantages, including the lack of animal pathogens such as prions and viruses, low cost and the large-scale production of safe and biologically active valuable recombinant proteins, the case of scale-up, efficient harvesting and storage possibilities. However, N-linked glycans from plants differ from those of mammalian cells. For example in plants, beta-(1,2)-xylose residues have been shown to be linked to the core Man3GlucNAc2-Asn of glycans, whereas they are not detected on mammalian glycans, where sialic acid residues and terminal beta(1,4)-galactosyl structures occur instead. The unique N-glycans added by plants could impact on both immunogenicity and functional activity of the protein and, consequently, may represent a limitation for plants to be used as a protein production platform. Indeed, the immunogenicity of beta-1,2-xylose residues in mammals has been described in for example Jin et al. (2006) Glycobiology 16: 349-357.
The enzyme that catalyses the transfer of xylose from UDP-xylose to the core β-linked mannose of protein-bound N-glycans is beta-1,2-xylosyltransferase (“XylT”, EC 2.4.2.38). The beta-1,2-xyosytransferase is an enzyme unique to plants and some non-vertebrate animal species and does not occur in human beings or in other vertebrates.
WO2007107296 describes the identification and cloning of beta-1,2 xylosyltransferases from the genus Nicotiana such as Nicotiana benthamiana. Various strategies have been applied to avoid beta-1,2-xyosyl structures on glycoproteins produced by plants. WO2009056155 describes an RNA interference strategy for the generation of Nicotiana benthamiana plants which are deficient in the formation of beta-1,2-xyosyl structures as well as devoid of alfa-1,3-fucosyl structures on heterologous glycoproteins.
The cleanest approach for the production of glycoproteins lacking xylosyl-epitopes in Nicotiana benthamiana would be the generation of a full knock-out of the beta-1,2-xylosyltransferase gene in this plant. The latter is however not a straight-forward strategy because of the documented presence of at least two beta-1,2-xylosyltransferases (see WO2007107296) and the extremely low efficiency of homologous recombination in plants. Another strategy would be the generation of null mutations in all of the functional alleles of the genes possessing beta-1,2 xylosyltransferase activity in Nicotiana benthamiana. Plant populations mutagenized by ethyl methanesulfonate (EMS) have proved invaluable to plant biologists as a means of dissecting genomic traits. Nicotiana benthamiana is however a higher plant and is estimated to contain 30.000 to 50.000. A major obstacle in Nicotiana benthamiana genetics is the lack of large mutant populations required for mutant gene identification. Such a useful N. benthamiana population would ideally contain at least one mutant allele for every N. benthamiana gene. Mutant N. benthamiana plants can be produced through the use of DNA damaging agents such as EMS, X-rays, or fast-neutrons. However, no stocks of mutagenized M2 seeds, originating from a large population of M1 plants, are available for screening mutations in candidate genes. The aim of our research was to provide a mutant population of N. benthamiana, to screen for null alleles in said population for genes that encode beta-1,2-xylosyltransferase activity with the ultimate goal to evaluate the possibility of obtaining an induced mutant plant completely deficient in the xylosyltransferase activity.